The objective of this project is to define the reaction mechanisms of human type I 3beta-hydroxysteroid dehydrogenase/steroid 5-4-ene-isomerase (3beta-HSD/isomerase) by relating function to structure. In placenta, type I 3beta-HSD/isomerase catalyzes the conversion of maternal pregnenolone to progesterone, a hormone that promotes uterine quiescence during pregnancy. The enzyme also competitively utilizes dehydroepiandrosterone, the primary steroid product of the fetal adrenal gland near term, to produce androstenedione that is further metabolized to l7beta-estradiol. Thus, placental 3beta-HSD/isomerase bridges hormonal communication between the mother and fetus to mediate the locally increased estrogen/progesterone balance that has been associated with the onset of labor. The type I 3beta-HSD/isomerase is also the dominant form in the mammary gland and breast tumors. The cDNA for the type I enzyme has been over-expressed by baculovirus in insect cells. The substrate domain was localized by affinity radiolabeled peptide studies. The following studies test our central hypothesis: The 3beta-HSD and isomerase activities are catalyzed at overlapping, but distinct, catalytic sites. When NAD+ is reduced to NADH by the 3beta-HSD activity, the NADH produced activates the isomerase reaction by inducing a conformational change in the enzyme protein. Thus, the utilization of coenzyme at a single domain bridges the sequential reactions. Multiple mutations of Tyr and Lys in the YXXXK motifs and of targeted Tyr and Asp residues determine if these amino acids catalyze the 3beta-HSD and isomerase mechanisms, respectively. The cofactor site responsible for NAD+ reduction is localized using the 3beta-HSD-site-directed, affinity labeling cofactor analog, 5'-(p-fluorosulfonyl[14C]benzoyl)adenosine. The isomerase-activator site is identified using the NADH-site-directed analog, 8-[(4-bromo-2,3-[3H]dioxobutyl)-thio]-adenosine 5'-diphosphate. A "cross-over" study using both affinity alkylating nucleotides definitively tests our hypothesis of a single coenzyme domain. Specific mutations are introduced in the identified coenzyme domain(s) followed by baculovirus- expression of the mutant enzymes in insect cells. Kinetic and pH- dependence studies on the purified mutants characterize the amino acids responsible for substrate and cofactor utilization to define the multiple reaction mechanisms. The deletion of a membrane-spanning domain produced a fully active, cytosolic enzyme that can be purified without using detergents and appears to be identical to the microsomal wild-type enzyme. The kinetic properties of the purified cytosolic enzyme are compared to those of the purified wild-type enzyme. The absence of detergent in the pure cytosolic enzyme preparation facilitates the growth of crystals for future structural studies.